Field of the Invention
The present invention relates to light microscopy-based chips and quantitative analysis methodology for localized surface plasmon resonance (LSPR) biosensing and imaging.
Description of the Prior Art
Localized surface plasmon resonance (LSPR) is an emerging technique in the field of label-free biosensing which is currently dominated by the closely related, but more mature surface plasmon resonance (SPR) technique. (P. Englebienne, Analyst, 123, 1599-1603 (1998); A. J. Haes et al., J. Am. Chem. Soc., 124, 10596-10604 (2002); N. Nath et al., Anal. Chem., 74, 504-509 (2002); B. Sepulveda et al., Nano Today, 4, 244-251 (2009); J. Zhao et al., Nanomedicine, 1, 219-228 (2006)). Both employ the coupling of light with metallic structures for the excitation of a plasmonic resonance and both take advantage of the fact that the resonance is sensitive to changes in the index of refraction near the metallic surface and thereby can be used to detect the presence of analytes such as proteins or nucleic acids. In SPR, total internally reflected light, typically introduced by a prism, is incident at the “resonant” angle that excites surface plasmon polaritons propagating laterally along a planar, thin metal film. The sensitivity of the resonance to the presence of analytes extends hundreds of nanometers above the thin film's surface. (L. S. Jung et al., Langmuir, 14, 5636-5648 (1998) and K. Kurosawa et al., Phys. Rev. B, 33, 789-798 (1986)). By contrast, the localized nature of the LSPR nanostructures, typically 50 to 150 nm in diameter, allows for a range of incident light angles to be utilized, from normal to totalinternally reflected, and the sensitivity to analyte is confined to within tens of nanometers from the surface. (M. D. Malinsky et al., J. Am. Chem. Soc., 123, 1471-1482 (2001)).
Both techniques can be used for imaging, such that spatial and temporal information of analyte binding is acquired, although the concept of LSPR imaging offers some distinct advantages over that of SPR imaging. First, because the spatial resolution of LSPR is restricted only by the size of the nanoparticle, the imagery is in principle diffraction limited and indeed spectroscopic-based biosensing with single nanostructures has already been achieved. (K. M. Mayer et al., Nanotechnology, 21 (2010) and G. J. Nusz et al., Anal. Chem., 30, 984-989 (2008)). By contrast, traditional SPR configurations have a lateral spatial resolution that is limited by the decay length of the surface plasmon polaritons, which is on the order of microns for the gold thin films typically employed. (C. E. Berger et al., Anal. Chem., 70, 703-706 (1998) and B. Rothenhausler et al., Nature, 332, 615-617 (1988)). In addition, the fact that SPR is sensitive to dielectric variations hundreds of nanometers above the metallic surface can result in a convolution of solution- and surface-based changes. Finally, the ability to excite LSPR resonances at a range of incidence angles allows for the straightforward incorporation into commercially available wide-field microscopes employing high numerical apertures whereas SPR imaging configurations must be custom built.
In order to realize its promise and overtake the older SPR technology, methodologies for LSPR biosensing must be developed that allow for quantitative determination of important physical quantities such as the fractional occupancy of receptor sites at the surface. Ideally these measurements would be made on a platform also capable of LSPR imaging so that both spatial and temporal information could be gained simultaneously. The fractional occupancy of binding sites, f, is of particular interest because it can be used to calculate the analyte concentration at the sensor surface if the reaction rate constants are known or, conversely, to determine the rate constants if the analyte concentration is known.